A great number of methods are known in the prior art for isolating nucleic acids, such as DNA and RNA, which allow an isolation from sample materials of quite various origin. These include sample materials of natural origin, for example complex samples that can be obtained from humans, animals, plants and the environment, and sample materials from a laboratory culture, such as microorganisms and cell culture materials.
The fundamental principle of nucleic acid isolation is generally independent of the type of sample material and can be divided into a few steps: an optional digestion of the sample material, for example in form of a lysis, the actual nucleic acid isolation and optionally the purification of the isolated nucleic acids. The isolation of the target nucleic acids from the contaminating sample constituents such as for example cell fragments and molecules different from nucleic acids, as well as the purification of the nucleic acids can be divided into two types of methods, which differ mainly in the number of separation steps and the separating agent:
In a classical method, often a chaotropic agent-containing aqueous buffer is first added to the sample material for lysis of the cells respectively digestion of the sample, said buffer usually containing a guanidinium salt. This is often followed by the denaturation of contaminating proteins and extraction of the nucleic acids by mixing with an organic extractant, which usually contains phenol and/or chloroform. After separating the nucleic acid-containing aqueous phase from the organic phase, for example an alcohol precipitation is carried out to purify the nucleic acids from water-soluble contaminants and phenol/chloroform components.
This extraction method has the disadvantage that a small portion of the organic extractant may always remain in the aqueous phase, so that several time-consuming steps of purification of the nucleic acid phase are required, which have an adverse influence on the yield of the target nucleic acids. Even after several purification steps, the purified nucleic acid samples often do not have the necessary purity for subsequent applications, in particular with regard to residuals of the extractant phenol.
An alternative multi-step method avoids the use of toxic organic solvents by using a solid mineral carrier material, which is based for example on a silicon dioxide compound. Owing to the generally mainly selective binding of the nucleic acids to the carrier material, the nucleic acids are isolated in the form of nucleic acid-carrier material complexes. The initial step in this method is usually also cell lysis, unless the nucleic acids to be isolated are already present in free form. For degrading cell constituents and/or other sample constituents, for example proteins, usually a chaotropic agent-containing aqueous buffer and usually a protein-degrading enzyme are added to the sample material. The nucleic acids released from the used sample material are then contacted with the carrier material under suitable binding conditions, usually in the presence of alcohol. The first required isolation step is usually based on selective adsorption of the nucleic acids to the carrier material, usually followed by a separation of the resulting nucleic acid-carrier material complexes from the surrounding liquid. This can either take place almost simultaneously in a filtration process, if the carrier material functions for example as filter matrix, or in a subsequent step for separating the complexes from the suspension. Optionally this is followed by a purification of the bound nucleic acids in one or more washing steps. Usually an elution follows as a final step of the isolation process, in which the bound nucleic acids are released from the carrier material with an aqueous reagent. This step is optional, since in some subsequent applications, for example the polymerase chain reaction, the binding of the carrier materials to the target nucleic acids is not disturbing. This multi step isolation method has become established owing to its advantages, such as improved yield and purity of the nucleic acids, compared to other classical methods.
A number of similar isolation methods that are based on the multi-step method described above are described in the prior art (see for example U.S. Pat. No. 5,234,809, WO 93/11221, WO 95/01359 and WO 2006/084753), which are characterized for example by the use of different carrier materials, different mechanisms for separating the nucleic acid-carrier material complexes from the sample and/or different reagent compositions.
Furthermore, WO 2009/144182 (Fabis et al.) discloses a universal composition for a lysis, binding and washing reagent for the isolation and purification of nucleic acids, which comprises at least one chaotropic compound, at least one buffer compound and at least one non-ionic surfactant. This reagent has various advantages, as it is more stable in storage than the Tween-containing formulations known in the prior art, used for example for cell lysis, as it increases the nucleic acid yield and/or produces nucleic acid eluates without turbidity.
All manual or automated methods known in the prior art, including the aforementioned examples, show a continuous further development of the isolation and purification of nucleic acids from various sample materials, among other things for increasing yield, purity, speed and/or sample throughput. The sample materials used in the prior art generally have in common that they contain the nucleic acids to be isolated in quite large amounts and/or they are similar with respect to their nature and/or composition, that the type of sample material has little if any influence on the efficiency of nucleic acid purification. Furthermore, the methods known in the prior art are as a rule also optimized in relation to one sample material or to similar sample materials (for example blood and blood products). However, the methods known in the prior art are as a rule not designed for and/or are not capable of also quantitatively purifying small amounts of nucleic acids from very different sample materials, which for example differ considerably with respect to their pH, their composition, their protein content and in particular their salt content, in high purity and for a high sample throughput. In particular the known methods are not intended for purifying nucleic acids quantitatively from bioprocess samples, such as for example aqueous buffer solutions with different compositions. Such a method would be of interest for example in the area of the production of biotechnological products, in particular for biopharmaceuticals.
Biopharmaceuticals include in particular proteins and peptides (for example antibodies, antigens, growth hormones), nucleic acids (for example DNA, RNA, plasmids, siRNA), viruses and/or vaccines. Biopharmaceuticals are produced using the means of modern biotechnology, in particular with the aid of natural or genetically modified organisms. Production usually employs eukaryotic or prokaryotic host cells, which are often modified by genetic engineering so that they are able to produce the desired biopharmaceutical. Respective genetic engineering techniques are well known in the prior art and include but are not limited to recombinantly introducing a gene encoding the product of interest such as e.g. a protein into a cell. For this purpose, a vector may be used. The respectively modified cell is then capable of producing the product of interest. In particular, for this it is possible to use cell lines from mammals and insects, recombinant microorganisms in bacterial or yeast cultures, viruses, as well as plant meristems and genetically modified plants. Biopharmaceuticals are applied inter alia for the prevention, diagnosis and treatment of diseases. Furthermore, biotechnological products, including biopharmaceuticals can also be produced by biotransformation. For this purpose, genetically modified or unmodified cells, such as in particular bacteria or yeast cells, are contacted with a chemical precursor and the cells transform said precursor to a desired product using their enzymatic machinery.
In the prevention of diseases, various immunization techniques for the prevention of bacterial and viral diseases have become established, and thereby biotechnologically produced vaccines are becoming increasingly important. These include viruses, antigenic components of viral envelopes in conjugated or unconjugated form, or pure DNA components of viruses, bacteria or other organisms. Parts of a viral envelope or recombinant viruses can also be used for preventive vaccination against diseases that are caused by a viral infection. For example, this technique are applied for HPV (human papillomavirus) vaccination for preventing cervical cancer. Mainly monoclonal antibodies are applied for molecular diagnostics, and are used for example for immunophenotyping, immunohistological determinations or ELISA. Treatment of diseases is another constantly growing field of application for biopharmaceuticals. Treatments are carried out using recombinant proteins, hormones, recombinant organisms, viruses, recombinant immune cells or also using genes.
Biotechnological products, such as biopharmaceuticals in particular, usually go through a multistage purification process. To obtain the biotechnological product, the producing host may for example be lysed, or the biotechnological product can be isolated from the culture supernatant. Purification of the biotechnological product from the culture supernatant and/or the lysate is generally carried out using chromatographic methods. The purpose of this purification is usually to remove contaminants, for example cell fragments, cellular molecules, culture medium and salts from the resultant biotechnological product and to enrich it. Moreover, especially in the case of biopharmaceuticals, it is of particular interest to achieve a considerable reduction in the content of nucleic acid contaminants (in particular host cell nucleic acids), as these contaminants may, for example depending on the concentration and the host, impart a potential oncogenic risk to the final biopharmaceutical.
Usually, for purification of biotechnological products, such as biopharmaceuticals in particular, purification steps are applied by means of standard chromatographic techniques, for example ion exchange chromatography, protein A/G affinity chromatography, hydrophobic interaction chromatography and/or hydrophilic interaction chromatography, which are generally selected specifically for the biopharmaceutical agent to be purified in each case. The eluates from these purification methods, which are here preferably encompassed by the term bioprocess samples, are as a rule aqueous purification fractions, corresponding to these purification methods. Depending on the selected chromatographic technique, they are usually characterized by a wide range of different compositions of ingredients, for example with respect to the purification buffer used (for example acetate, phosphate, citrate or maleate buffer), to the salt concentrations, pH values, the phosphate components and/or the protein content. Bioprocess samples of successive purification steps usually display an increasing degree of purity of the biotechnological product, for example of the biopharmaceutical, which as the end product of the purification process must fulfil the purity criteria of regulatory authorities, so that it can be authorized for example as a medicinal product or diagnostic product.
A number of quality guidelines for medicinal product testing and authorization for the use on human beings are issued by national and international authorities. These include the ICH guidelines (EU, USA, Japan), the EMEA guidelines (EU) and the FDA guidelines (USA) and WHO guidelines. These guidelines define, for medicinal products, among other things the maximum permissible residual amounts of various molecules that originate from host organisms, including the amount of endotoxins, the amount of proteins and the permissible amount of prokaryotic or eukaryotic DNA. With respect to the DNA of the host organisms, according to the WHO decision of 1987, an amount of 100 pg per dose of medicinal product to be administered must not be exceeded. Ideally, and advised by the FDA, a much lower residual amount of host DNA of only 10 pg at most per dose to be administered is to be achieved, to ensure greater safety for the patient.
Similar rules also apply to other biotechnology products, for which nucleic acid contaminations, for example with viral nucleic acids, can represent a problem.
A trend that is seen in the determination of these nucleic acid contaminants is the use of very sensitive molecular detection techniques, for example the quantitative polymerase chain reaction (qPCR). Corresponding methods are known in the prior art and form the subject matter of various patent applications (see for example US2009/325175). However, to perform said detection it is first necessary to isolate effectively the small amounts of contaminating nucleic acids (for example from the host) from the bioprocess samples. This use is not only of interest for nucleic acid analysis in the finally purified end product, it is in particular also of great importance for the purification fractions arising continuously one after another in the purification process, as analysis of these purification fractions enables monitoring and assessing the successful outcome of purification. In this way, it is also possible to detect faults in the purification process more quickly. Therefore it is common in the prior art, at various points of the purification process and preferably at each stage of the purification process, to determine the amount of target nucleic acid (for example of a contamination, in particular with host DNA) in the respective purification fraction. This use imposes particular challenges on the nucleic acid purification method to be used, because—as explained—the various purification fractions, as a rule differ considerably from each other with respect to their chemical composition and their pH. Here, owing to the high sample throughput, it is desirable, despite the very different sample materials, to be able to use a uniform method of nucleic acid purification, that can ideally be automated, and by which even very small amounts of nucleic acids can be isolated as quantitatively and consistently as possible. Methods for isolation of nucleic acids being commercially available for this purpose so far do not fulfil these requirements satisfactorily, as they have various disadvantages.
The DNA Extractor Kit from the company WAKO (#295-50201) is a manual method, in which contaminating proteins are denatured by the use of a chaotropic salt (NaI) and an anionic detergent (sodium N-lauryl-sarcosine). After adding alcohol and glycogen, the nucleic acids in the sample are precipitated, wherein the precipitate can also contain entrained contaminants, which can be disturbing in a subsequent PCR reaction. Furthermore, the method is time-consuming and cannot easily be automated.
The PrepSEQ Residual DNA Sample Preparation Kit from the company ABI is a method that uses magnetic silica particles and is suitable for automation. A disadvantage of this method is that the sample material first requires laborious preparation, in that the samples must be treated individually in the sequence 1.) adjustment to an optimum pH and 2.) adjustment of NaCl concentration in the sample to >0.5 M. This preparation is necessary because of the varying nature/composition of the bioprocess samples, as otherwise the subsequent isolation operation, in particular with small amounts of nucleic acids in the sample material, would not function efficiently. This preparation is time-consuming, because often it can only be done manually, so that this method does not permit high sample throughput. Automation of subsequent steps of nucleic acid isolation would not be useful in this case, as the step of individual sample preparation would still be included.
Other methods, which are based on the automated use of magnetic particles, are for example the methods of isolating nucleic acids with the laboratory automate QIAsymphony using the correspondingQIAsymphony Kits from the company QIAGEN GmBH. These applications enable isolating and purify nucleic acids quantitatively from a number of different sample types such as tissue, whole blood, plasma, serum, urine, forensic sample materials etc., but they are not originally designed to fulfil the special requirements on sample materials such as bioprocess samples and in particular purification fractions. The special nature of these samples arises, as explained, from the use of a large number of specific chromatographic techniques, which often result in aqueous bioprocess samples with very different chemical composition and different pH values, which contain inter alia inhibitors for known methods of nucleic acid isolation and purification. Furthermore, generally they only contain small amounts of the target nucleic acid (for example host DNA).
The disadvantages of the commercially available methods of nucleic acid isolation, including the methods given above as examples, emphasize the existing demand in the pharmaceutical and biotechnology industry for a suitable method for isolating nucleic acids, which allows even small amounts of nucleic acids, such as for example contaminating nucleic acids (which originate for example from host organisms or host cells) to be purified, wherein the method should preferably represent a quantitative, consistent solution that can be automated.
Besides the pharmaceutical and biotechnology industry, there are yet other fields of application in which detection of nucleic acids that are present at very low concentrations, in various sample materials, is of considerable interest. These include molecular medical diagnostics, in which it is for example of interest to detect, from body materials, foreign nucleic acids, such as fetal, viral, and microbial nucleic acids, which are present in small amounts. Obtaining freely circulating fetal nucleic acid from maternal blood samples is often crucially important in prenatal diagnostics, as it represents a genetic method for analysing the fetus, which does not require amniocentesis and therefore holds no risk for the unborn offspring. In the diagnosis of infections, the isolation of microbial nucleic acids that are present in small amounts provides early evidence about an infection and permits early treatment of the patient. Furthermore, if there is a risk of infection, rules of conduct can be apprehended early, for example temporary quarantine of the patient, in order to limit or even prevent spread of the infection. Very early pathogen detection is of great importance especially in the case of aggressive, highly infectious pathogens, which even in very small numbers cause an infection with a serious and barely treatable course of disease. This applies, for example, to Shiga toxin producing Escherichia coli (STEC) bacteria, infection with which leads not infrequently to the death of the patient, as a classical antibiotic therapy cannot be used. Early recognition of the infection, as well as treatment and early isolation of the patient, can save lives. Endogenous nucleic acids that occur in small amounts are also of interest in molecular diagnostics. These include for example small non-coding RNAs such as in particular microRNA (miRNA) molecules, which regulate gene expression at the posttranscriptional level. They are used for example as marker molecules in the area of oncological diagnostics.
Forensics is another field of application in which nucleic acid diagnostics plays an important role, because often only that way the decisive evidence for clarifying forensic questions can be provided. Forensic sample materials are generally characterized by an especially wide variety, which in principle encompasses all materials and fluids that can carry or contain human nucleic acids. In particular, however, they are sample materials that often only contain very small traces of the nucleic acids to be detected. A molecular detection system therefore requires a very efficient and effective method of isolating nucleic acids, in which there are only minimal or ideally no losses of the nucleic acids that are only present in small amounts, in order to enable subsequent detection at all.
Molecular nucleic acid detections are also very important in food and environmental diagnostics, for example for detecting contaminations by bacteria, viruses, fungi and/or protozoa in the area of food production, foodstuff inspection, breeding and monitoring of plant developments and/or also in the analysis of water, soil and air quality. Also in this case the sample materials can have a very diverging composition and may only contain very small amounts of the nucleic acids of interest. Molecular detection based on microbial nucleic acids, instead of a method of detection by means of cultures of microorganisms, is a method that is conceivably more time-saving and more specific.
In basic research, synthetically produced nucleic acids, for example small interfering RNA (siRNA), are used in genome analysis. The chemical production of nucleic acids requires purification processes that are generally carried out by means of chromatographic techniques. Also in this case, a molecular method of nucleic acid isolation and/or purification would be of interest, which permits a quantitative isolation of the nucleic acids and allows to obtain the product in a low-salt solution. Consequently here as well, the composition of the purification fractions must not have any disturbing effect on the method of nucleic acid isolation and/or purification.
The present invention is therefore based among other things on the problem of providing a method of isolation and/or purification of nucleic acids that are present in small amounts in sample materials of varying composition, which in particular permits the quantitative detection of the isolated nucleic acids. Moreover, in particular a problem to be solved by the present invention is to provide a respective method, which enables the preferably quantitative isolation of nucleic acids from various bioprocess samples, in particular from various purification fractions obtained in a chromatographic purification process, even if these only contain a small amount of the nucleic acids to be isolated.